The Influence To Enzyme Production And Properties Of S7-xyn And RML By Fusion To Cellulose Binding Domain

Cellulose binding domain (CBD) belongs to one of important family members ofcarbohydrate-binding modules (CBM). Most hemicellulose and cellulose degrading enzymeshave modular structures consisting of usually a catalytic module and one or more noncatalyticCBD which are often separated by a linker segment. The unique biological role of CBDappears to mediate tight association between the enzyme and the substrate. This can increasethe effective enzyme concentration on the substrate, thereby enhancing the hydrolytic action.Moreover, Some CBDs can be used as a secretion enhancer for secretory production ofheterologous protein in Pichia pastoris. In order to solve the main obstacles of xylanasebleaching treatment in the pulp industry, such as poor thermal stability andHalkali resistanceH,It’s very necessary to acqure a xylanase which is not only active at high temperature andunder alkaline conditions, but has efficient cellulose adsorption and hydrolytic enzymeactivity. What’s more, it can also provide some solutions when RML is used in industrialproduction with high cost and low enzyme activity. In this study, different CBDs wererespectively fused to the B.halodurans S7xylanase and RML in various ways. Then theexpression cassettes for the fusion proteins were constructed and expressed in P. pastoris.Compared to native enzyme, the catalytic and physicochemical properties of the chimeraswere characterized. Finally the recombinant xylanases were used to evalutate the biobleachingpotential on reed pulp and compared with or without S7-xyn. The specific research content islist below.(1) Recombinant xylanases expression in Pichia pastorisFour recombinant enzymes (CES7, S7CC, CES7CCand S7CCCC) were produced by P.pastoris. When the fusion gene of CES7was expressed in P. pastoris, the secretion efficiencywas improved significantly, compared with the native S7xylanase. The amount of secretedxylanase activity as CES7(144U/mL) was increased about30%than that for S7xylanase(110U/mL) at120h induced by methnol. However, the amount of secreted xylanase activityas S7CC(71U/mL) was reduced by35.4%. What’s more, the xylanase activities of CES7CC(14U/mL) and S7CCCC(11U/mL) were remarkably reduced by87%and90%than that for S7xylanase. It is indicated that single CBMEndIIcan enhance the enzyme activity of S7-xyn,CBMCelAhad aHinbibitionalH HeffectHfor S7-xyn.(2) Cellulose-binding studyThe binding capabilities of the chimeras were investigated on microcrystalline celluloseand three kinds of pulps (Reed pulp, bamboo pulp and wheat steaw pulp). The percentages ofadsorption toward these substrates were all enhanced significantly for the chimeras comparedwith native enzyme S7-xyn. The amount of S7CCbound was remarkably higher than in thecase of CES7. What’s more, the chimeras with multiple CBMs, CES7CCand S7CCCC, had astronger binding capability than others, no matter what the substrates were. In addition, whendifferent substrates were used to analyze the bind capability of these chimeras, the case ofpulps is better than microcrystalline cellulose, although the situation of each pulp also is notidentical.(3) Effects of pH and temperature on the activity and stability of the chimeric xylanasesThe chimeric enzymes displayed an optimum pH of9.0except S7CC, which had anoptimum pH of10.0. What’s more, the chimeric enzymes had a broad pH range frompH6.0-10.0other than CES7CCand S7CCCC. Temperature optimum measurement hasdemonstrated that maximal activity occurred at75oC but not CES7and S7CC. Compared withthe mature S7xylanase, the chimeric enzymes had the similiar characteristics as the matureenzyme. The thermal stability of the enzyme was determined at65oC. The enzymes (S7-xyn,CES7, S7CC, CES7CCand S7CCCC) retained63.2%,69%,45.8%,100%and75.6%after4hincubation respectively.(4) Recombinant xylanases treatment of reed pulp and ECF BleachingAt the same bleaching reagent consumption, S7CCCC-treated pulps have about1.8%and3.8%ISO higher brightness than the S7-xyn-treated pulps and the control DQP-treated pulps.Other three enzymes treatment enhanced brightness than the S7-xyn-treated pulps in a certainextent, respectively. What’s more, the kappa numbers decreased separately after bleachingwith pretreatment of these enzymes. Silimiar phenomenon was observed that thedeligification abilities of enzymes which were fused by CBM are all stronger than S7-xyn.This result shows that the chimeric xylanases have a good application prospect in thebleaching of pulp industry. (5) Fusion protein CRML expression in Pichia pastorisA fusion protein with a CBD has been successfully constructed and expressed in Pichiapastoris. Compared with that of the mature RML without CBMCelA-fusion, secretion ofCBMCelA-RML by this fusion construct was about17%enhanced in addition to similartemperature activity and stability between RML and CBMCelA-RML. Therefore, the CBMCelAfrom THEGII can be used as a secretion enhancer for secretory production of heterologousprotein in Pichia pastoris.By the research of the recombinant xylanase and RML, It’s possible to realize not only theapplication of new functional xylanases in the pulp bleaching industry, but also improve theRML enzyme activity and reduce the cost of RML production in order to more effectiveapplication in industry.